Western Blotting
Assembling the Cassette Materials *Western Transfer **Methanol **Western Transfer buffer **Sponges X2 **Blotting Paper X2 **PVDF membrane **SDS-PAGE Gel **Plastic Cassette *Blocking **PVDF membrane from Western Transfer **Skim milk powder **1X PBST *Ab Probing **Primary antibody aliquot (1° Ab) **Probing tray **PVDF membrane from 2° Ab probing **Skim milk powder **1X PBST *Detection on Film **ECL Plus/Prime or Millipore Crescendo/Forte Detection Kit **X-ray Film Method ''Assembling the Cassette #Open the plastic cassette with the black side down. #Activate the PVDF membrane by removing it from its protective covers and soaking it in methanol for 2 minutes. After this, equilibrate the activated PVDF membrane in Western Transfer Buffer for 5 minutes. ''The methanol can be reused numerous times. #Place sponges in Western Transfer Buffer until they are soaked well and then place one piece of sponge on the black side of the plastic cassette. #Soak blotting papers in Western Transfer Buffer and then place one blotting paper on top of the wet sponge (on the cassette). #Carefully remove the gel and place it over the blotting paper. Pour some Western Transfer Buffer over the gel and immediately place the PVDF membrane over it. #You may nick one corner of the gel as well as the PVDF membrane so ensure correct orientation when immunoblotting the PVDF membrane. A plastic pipette (''1 mL) can be used to smooth any bubbles between the gel and PVDF membrane interface.'' #Overlay the PVDF membrane with blotting paper and then with another sponge. Close over the white side of plastic cassette ad engage the lock on the side. ''Running the Western Transfer #Place the plastic cassette in the mini tank. Put a stir bar and an ice packet on the side. Then add transfer buffer up to where the mini tank curves. There are holes and the buffer can spill. ''The transfer proceeds from the black side (SDS-PAGE Gel) → white side (PVDF membrane). Ensure that electrodes are connected to proper ends of the Cassette Holder: Black (Negative) and Red (Positive). Add the ice bucket to the side or place the entire apparatus at 4 °C. You can also drop a stir bar and place over a stir plate for more efficient buffer mixing and cooling. #The transfer goes at 350 mAmps for 1 hour or at 300 mAmps for 1 hour 15 minutes. #After the Western transfer is finished, take out the cassette and place it in transfer buffer. Remove the top sponge and peel off the top blotting paper. #Cut off any extra membrane. If a protein marker was used, check that the marker has transferred to membrane. #Place the PVDF membrane in 1xPBST buffer and rock for 5 min at room temperature. Repeat one more time. #Block the PVDF membrane with 5% skim milk for 2-3 hours at room temperature. ''Primary Antibody Probing ''Ensure that the PVDF membrane is not dry at any time during the probing. Remove bubbles as best you can as they impair the antibody binding. #Dilute 1° Ab to appropriate dilution. For FLAG M2 monoclonal use 1:10,000 dilution (1 µL of Ab in 10 ml of 3% skim milk in PBST). #Place the PVDF membrane in a probing tray and pour in the 1° Ab until the membrane is completely submerged. #Place the tray on a rocker at 4 C and allow 1° Ab probing to go ON. If you are unable to wait overnight you can try probing at room temperature for 1 hour. This might result in higher background though. ''Secondary Antibody Probing #Pipette out the 1° Ab and place back in the tube. They are reusable. #Wash 4X in 20 mL of PBST. Place over agitator at room temperature at medium speed setting for 15 minutes to give a vigorous wash. After the 4th wash do not throw out the PBST to prevent drying of blot. #Dilute 2° Ab to appropriate dilution. For α-FLAG mouse IgG use 1:10,000 dilution (1 µL of Ab in 10 mL of 5% skim milk in PBST). #Pour out the 4th PBST wash and immediately pipette in the 2° Ab to the PVDF membrane. #Place on rocker at room temperature for 1 hour. #Wash 4X in 20 mL of PBST for 15 min at room temperature. Detection on Film ''Remove the Detection kits prior to detection to equilibrate reagents to room temperature. #After starting 4th wash with PBST, prepare detection solutions as per manufacturer protocol. For ECL Plus Solutions A and B are mixed in a ratio of 50:1 and for ECL Prime, A and B are mixed in a ratio of 1:1. Millipor kits are single solutions only. #Drain excess PBST from membrane and place protein side up on a sheet of SaranWrap. Pipette the mixed detection reagent on to the membrane and incubate for 5 minutes at room temperature. #Drain off excess detection reagent by holding the membrane gently with forceps and touching the edge against a tissue. Place the blots protein side down on to a fresh piece of plastic wrap, wrap up the blots and gently smooth out any air bubbles. #Place the wrapped blots, protein side up, in an X-ray film cassette. Tape it down so that you can match the film and the blots after the film has been developed to spot the markers. Ensure there is no free detection agent in the cassette; the film must not get wet. #Take the film cassette (and timer) to the dark room and turn on safety light. #Place a sheet of X-ray film on top of the wrapped membrane. Close the cassette and expose for 15 seconds. Open the cassette and flip the sheet of film and then expose for 1 minute exposure. Time of exposure will depend on each occasion. #When doing an exposure, bend one corner of the film towards you so that you can identify the positions of the membrane and match the markers. #After each exposure immediately process the film. Place the film with one side aligned with the feed side of the machine. The machine roller will pull in the film slowly. After you hear the beep, wait 10 seconds to turn on the light. Be sure to put away unused films back in their holder before turning on the light. #After processing the films, match the film with the membranes and mark the markers.